Mononucleotide run sequence slippage sequencing problems
Identification of DNA Sequence Slippages
- The trace data becomes mixed after a long mononucleotide (single base) run in the DNA template (Figures 1 and 2).
- Can also occur on long dinucleotide repeats in a manner analogous to stutter bands that occur in microsatellite polymerase chain reaction (PCR) amplification.
Figure 1. Sequence slippage on a mononucleotide T run.
Figure 2. Sequence slippage on a mononucleotide C run.
Causes of DNA Sequence Slippage Problems
- Long runs of a mono nucleotide base causes the DNA polymerase to "slip" on the template.This occurs by either the template or extension product looping out and rehybridizing and resulting in the generation of sequencing products of varying size. These sequencing products then appears as mixed signal in the trace downstream of the mono nucleotide run.
- Insertion and deletions in one allele. This can occur when two alleles are sequenced together (PCR templates) and one of the two mononucleotide runs has an InDel polymorphism.
Solving DNA Sequence Slippage Problems
- Sequence the DNA template from both directions. For example, if the template shows slippage after using the forward primer then sequence the template from the reverse direction. You may need to make a custom primers to do this if the template is large.
- Use a custom primer designed to hybridize just outside the mononucleotide or dinucleotide run region.
- Use the sequence-by-mutagenesis (SAM) approach to avoid have long mononucleotide runs in your templates.
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